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The Influence of Trichosanthin on the Induction of Ige Responses to Ovalbumin Under Adjuvant-Free Condition

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The Influence of Trichosanthin on the Induction of Ige Responses to Ovalbumin Under Adjuvant-Free Condition Cell Research(1995),5,69—74 The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant-free condition JI YONGYONG, CUIHONG Y ANG, M ING Y E H Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China. ABSTRACT Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL /6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovalbumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when OVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens. Key words: Trichosanthin, ovalbumin, allergen, IgE response. INTRODUCTION Trichosanthin (TCS) is an effective protein component of a Chinese herb medicine, which, has long been used in history for abortion induction[1]. Recently it has been found that TCS has anti-HIV effects[2], which is arousing much interest in it. Its amino acid sequence, DNA sequence, and 3D structure have all been worked out[3, 4]. In clinical application it occasionally induce anaphylaxis in certain patients[5]. Previously we had done a number of studies on the TCS specific IgE responses in mice immunized with TCS and adjuvant alum[6]. Since using the protein alone can induce anti-TCS IgE responses in human body, we intend now to study the TCS-IgE 1. The work is specifically dedicated to Prof. Zhen YAO for his 80-yeaxs birthday TCSon the induction of IgE responses without the influence of adjuvant in mice. That would be possibly more closer to the status happened in human body. Furthermore cross-reactivity of IgE antibodies in sera with different allergens in some of common allergenic determinants on different allergens is suggested to be the reason. However that it may be due to certain structural characteristics in antigen binding site of IgE molecules was also suggested[8]. And the possibility of allergens may influence different lymphocyte clones through mediators or cells that involved in the induction of IgE antibody responses within microenvironment could not be excluded also. In this paper we intend to establish a reproducible TCS-specific IgE antibody responses without the use of adjuvant to investigate its allergenic potential, and try to study the cross reactivity of allergen at cell clone level. MATERIALS AND METHODS Animals 1. Mice: C57BL/6J mice, female and 12 weeks of age 2. Rats: Wistar rats, female with 200g body weight. Reagents 1. Antigens 1) TCS: Crystalized TCS in Trichosanthin Injection Solution (TCS-J), produced by Jin-San Pharmaceutical Factory, 1.2 mg /ml, was used as immunogen. Refined TCS (TCS-W), produced by Wu-Han Biological Products Research Institute, 2mg /ampule was used in the passive cutaneous anaphylaxis(PCA) test as a challenger and in ELISA as a coating antigen. TCS-J has now been commonly used in clinics for a number of years. Considering TCS-J induced immune responses would be more closer to clinical status, we used it as the immunogen. However we used TCS-W as a challenger to detect the formed TCS-J-specific IgE reponses in mice. The reason is that the source of TCS-W is available and abundant in our hands. And we have intentionally run parallel experiments to compare the PCA results following TCS-J or TCS-W challenge. The results showed no differences (unpublished data). 2) Ovalbumin (OVA) , produced by Shanghai Dong-Feng Factory for Reagents. 2. Evan’s Blue, purchased from Shanghai Chemical Reagent Company. It was dissolved in phosphate buffered saline (PBS) and was adjusted to 1% concentration ready for use. Immunization The TCS-J or OVA were diluted or dissolved in PBS respectively and was each adjusted to 10 μg/ml and 20μg/ml. Each mouse was intraperitoneally injected with 0.5 ml antigen solution. The protocol of immunization was referred in below. Assays of the antibody specificity and titer 1. ELISA: Indirect ELISA was used to detect the specific antibody in antiserum or in hybridoma culture supernatant. The procedure were briefly as follow. TCS-W or OVA were used as the coating antigens. 1:50 diluted antiserum or hybridoma culture supernatants were used as the first antibody . Peroxidase labelled goat anti-mouse Ig (anti-MIg) or goat anti-mouse IgE (anti-MIgE) were used as the second antibody . Tetramethyl benezidine (TMB) and hydrogen peroxide were used as substrate. 2. PCA: The assay was used to detect the antigen-specific IgE antibody in antiserum or hy- bridoma culture supernatant. According to Ovary[9], the assay was carried out briefly as follows. 68 YY JI et al. On the shaved back of rats, 0.1 ml antiserum diluted to 1:10 with PBS or 0.1 ml of hybridoma culture supernatant were intradermally injected for priming. On the next day 2 mg TCS-W or 3 mg OVA dissolved in 1% Evan’s blue were intravenously injected into the tail vein of the rat. After 30 minutes blue patches appeared at the primed site were accounted as positive reaction, i.e. the presence of antigen-specific IgE in the test samples. Construction of hybridomas The primed lymphocytes were from mesenteric lymph node of mice immunized according to immunization protocol for Group B (see below). Five days before fusion the mice from Group B were boosted again. The tumor fusion partner was either SP2 /0 or NS-1 myeloma ceils. The fusion was carried out according to the routine method, including selection by HAT medium and screening. RESULTS Does minute amount of TCS alone without adjuvant could induce anti- TCS IgE responses ? The ELISA results of TCS specific serum titer from mice immunized ip with 5 μg TCS-J at 2-week interval are shown in Tab 1. It shows that all mice after two immu- nizations produced anti-TCS antibodies. And the antisera also contained anti-TCS IgE antibodies since PCA results indicated positive reaction. Tab 1. ELISA and PCA results of antisera* from C57BL/6J mice im- munized with TCS-J ELISA（ig）(OD±SD) PCA(IgE) Times of Days after immunization immunization 1** 2 3 Control 1 2 3 Control 1 7 0.12 0.12 0.13 0.26 ± 0.01 ± 0.01 ± 0.01 ± 0.15 14 0.16 0.21 0.63 _ _ + _ ± 0.01 ± 0.09 ± 0.02 2 7 0.75 0.75 0.71 + + + ± 0.01 ± 0.04 ± 0.01 14 0.84 0.85 0.77 + + + ± 0.01 ± 0.01 ± 0.05 3 7 0.97 0.98 0.99 + + + ± 0.03 ± 0.01 ± 0.03 14 1.00 1.03 1.00 + + + ± 0.02 ± 0.06 ± 0.02 * Antisera were diluted to 1:50 or 1:10 with PBS used as test samples in ELISA and PCA respectively. ** The numbers of mice. Does TCS effect the induction of OVA-specific IgE responses? Through immunization of different combination of TCS-J and OVA , the influence of TCS on the induction of OVA-specific IgE responses in C57BL /6J mice could be 69 TCSon the induction of IgE observed. The protocol for the combined immunization of TCS-J and OVA is shown in Tab 2. Tab 3 shows the experimental results. Results from Group F show that OVA alone could not induce IgE responses in mice. However, under the influence of TCS, it could induce OVA-specific IgE responses (Group B). But TCS effects only when it was used one day before OVA immunization. In case of TCS used five days before, it showed no promotion effects on the induction of OVA-specific IgE responses(Group A). On the other hand, either TCS and OVA given at the same time (Group C) or OVA given first and then immunization with TCS (Group D), none could induce OVA-specific IgE responses, although the anti-TCS IgE responses were all induced in these groups. These results have been repeatedly reproduced in separate experi- ments. Tab 2. Protocol of combined immunization of TCS-J and OVA Groups of Mice Time (Day) A B C D E F G 0 TCS* - - - - - - 4 - TCS - - - - - 5 OVA OVA OVA+TCC OVA TCS OVA - 6 - - - TCS - - - 19 TCS - - - - - - 21 - TCS - - - - - 22 OVA OVA OVA+TCS OVA TCS OVA - 23 - - - TCS - - - 35 Bleeding * The antigens used in immunozation. Tab 3. PCA results of antisera** from C57BL/ 6J mice immunized with TCS-J or OVA or both Groups of mice* Challenger (Antigen A B C D E F G TCS + + + + + - - OVA) - + - - - - - * The grouping of mice was shown on Tab 2. ** The antisera were diluted to 1:10 with PBS and used for intradermal sensitization of rats. Anti-OVA IgE and anti-TCS IgE are from separate Nones or from one clone? The hybridomas constructed by the fusion of mesenteric lymph node cells from Group B mice and myeloma cells were tested for their antibody specificities against TCS and OVA. Tab 4 shows the results of specific antibody titer of antiserum from 70 YY JI et al. lymphocyte donor. It can be seen that both antibodies against TCS and antibodies against OVA are present, and both contains TCS- and OVA-specific IgE, since the PCA tests are both positive. The specificities of the antibodies secreted by hybridomas in separate wells tested in two experiments are listed in Tab 5.
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